Lipolytic enzyme variant

ABSTRACT

The properties of a fungal lipolytic enzyme can be altered by substituting amino acid residues corresponding to certain specified amino acid residues in the  T. lanuginosus  lipase. The altered property may be, e.g., an increased thermostability, an altered pH dependence, or an altered substrate specificity.

FIELD OF THE INVENTION

[0001] The present invention relates to variants of fungal lipolyticenzymes, particularly variants with altered properties, and to methodsof producing such variants.

BACKGROUND OF THE INVENTION

[0002] It is known to use fungal lipolytic enzymes, e.g. the lipase fromThermomyces lanuginosus (synonym Humicola lanuginosa), for variousindustrial purposes, such as addition to detergents and in baking.

[0003] WO 92/05249, WO 9219726, WO 97/07202 and WO 0032758 disclosevariants of the T. lanuginosus (H. lanuginosa) lipase having alteredproperties.

SUMMARY OF THE INVENTION

[0004] The inventors have found that the properties of a fungallipolytic enzyme can be altered by substituting certain amino acidresidues.

[0005] Accordingly, the invention provides a variant of a parent fungallipolytic enzyme, which variant comprises substitution of one or morespecified amino acid residues. The invention also provides a method ofproducing a lipolytic enzyme variant comprising:

[0006] a) selecting a parent fungal lipolytic enzyme,

[0007] b) in the parent lipolytic enzyme substituting at least onespecified amino acid residue,

[0008] c) optionally, substituting one or more amino acids other thanb),

[0009] d) preparing the variant resulting from steps a)-c),

[0010] e) testing a property of the variant,

[0011] f) selecting a variant wherein the property is altered comparedto the parent lipolytic enzyme, and

[0012] g) producing the selected variant.

[0013] The specified amino acid residue corresponds to any of thefollowing amino acids in SEQ ID NO: 1:

[0014] Q15, Y16, A18, A19, A20, N25, N26, E43, V44, K46, A47, A49, L52,Y53, S54, G65, L67, A68, L69, T72, K74, L75, V77, S79, R81, S83, S85,W89, D96, L97, K98, E99, G106, C107, R108, G109, T123, L124, K127, E129,A131, V132, Y138, V140, L147, A150, T153, Y164, D165, D167, S170, Y171,G172, A173, P174, R175, V176, G177, R179, A182, Y194, H198, N200, P207,P208, R209, G212, S214, H215, S216, S217, P218, E219, Y220, K223, S224,D234, I235, K237, I238, D242 to A243, P250, P253, D254, I255, P256,Y261.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Parent Lipolytic Enzyme

[0016] The lipolytic enzyme to be used in the present invention isclassified in EC 3.1.1 Carboxylic Ester Hydrolases according to EnzymeNomenclature (available at http://www.chem.qmw.ac.uk/iubmb/enzyme). Thesubstrate specificity may include activities such as EC 3.1.1.3triacylglycerol lipase, EC 3.1.1.4 phospholipase A2, EC 3.1.1.5lysophospholipase, EC 3.1.1.26 galactolipase, EC 3.1.1.32 phospholipaseA1, EC 3.1.1.73 feruloyl esterase.

[0017] The parent lipolytic enzyme is fungal and has an amino acidsequence that can be aligned with SEQ ID NO: 1 which is the amino acidsequence shown in positions 1-269 of SEQ ID NO: 2 of U.S. Pat. No.5,869,438 for the lipase from Thermomyces lanuginosus (synonym Humicolalanuginosa), described in EP 258 068 and EP 305 216. The parentlipolytic enzyme may particularly have an amino acid sequence with atleast 50% homology with SEQ ID NO: 1. In addition to the lipase from T.lanuginosus, other examples are a lipase from Penicillium camembertii(P25234), lipase/phospholipase from Fusarium oxysporum (EP 130064, WO98/26057), lipase from F. heterosporum (R87979), lysophospholipase fromAspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A10-155493), lipase from A. oryzae (D85895), lipase/ferulic acid esterasefrom A. niger (Y09330), lipase/ferulic acid esterase from A. tubingensis(Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipasefrom A. niger (WO 98/31790), lipase from F. solanii having anisoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO96/18729).

[0018] Other examples are the Zygomycetes family of lipases comprisinglipases having at least 50% homology with the lipase of Rhizomucormiehei (P19515) having the sequence shown in SEQ ID NO: 2. This familyalso includes the lipases from Absidia reflexa, A. sporophora, A.corymbifera, A. blakesleeana, A. griseola (all described in WO 96/13578and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parenthesesindicate publication or accession to the EMBL, GenBank, GeneSeqp orSwiss-Prot databases.

[0019] Lipolytic Enzyme Variants

[0020] The lipolytic enzyme variant of the invention comprises one ormore substitutions of the specified amino acid residues. The totalnumber of such substitutions is typically not more than 10, e.g. one,two, three, four, five or six of said substitutions.

[0021] In addition, the lipolytic enzyme variant of the invention mayoptionally include other modifications of the parent enzyme, typicallynot more than 10, e.g. not more than 5 such modifications.

[0022] The variant generally has a homology with the parent lipolyticenzyme of at least 80%, e.g. at least 85%, typically at least 90% or atleast 95%.

[0023] Specific Substitutions

[0024] The variant may comprise substitutions corresponding to one ormore of the following in the T. lanuginosus lipase (SEQ ID NO: 1) withthe proviso that the amino acid of the parent enzyme is substituted withanother amino acid:

[0025] Q15 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0026] Y16 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0027] A18 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0028] A19 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0029] A20 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0030] N25 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0031] N26 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0032] E43 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0033] V44 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0034] K46 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0035] A47 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0036] A49 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0037] L52 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0038] Y53 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0039] S54 to A, C, D, E, F, G. H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0040] G65 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0041] L67 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0042] A68 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0043] L69 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0044] T72 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0045] K74 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0046] L75 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0047] V77 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0048] S79 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0049] R81 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0050] S83 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0051] S85 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0052] W89 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0053] D96 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0054] L97 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0055] K98 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0056] E99 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0057] G106 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0058] C107 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0059] R108 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0060] G109 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0061] T123 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0062] L124 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0063] K127 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0064] E129 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0065] A131 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0066] V132 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0067] Y138 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0068] V140 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0069] L147 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0070] A150 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0071] T153 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0072] Y164 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0073] D165 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0074] D167 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0075] S170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0076] Y171 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0077] G172 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0078] A173 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0079] P174 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0080] R175 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0081] V176 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0082] G177 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W.Y;

[0083] R179 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0084] A182 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0085] Y194 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0086] H198 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0087] N200 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0088] P207 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0089] P208 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0090] R209 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0091] G212 to A, C, D, E, F, G, H, 1, K, L, M, N, P, Q. R, S, T, V, W,Y;

[0092] S214 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0093] H215 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0094] S216 to A, C, D, E, F, G, H, 1, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0095] S217 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0096] P218 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0097] E219 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0098] Y220 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0099] K223 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0100] S224 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0101] D234 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0102] I235 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0103] K237 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0104] I238 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0105] D242 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0106] A243 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0107] P250 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0108] P253 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0109] D254 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0110] I255 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0111] P256 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y;

[0112] Y261 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,Y.

[0113] Altered Property

[0114] The variant of the invention has one or more altered propertiescompared to the parent enzyme. The altered property may be, e.g., anincreased thermostability, an altered pH dependence, or an alteredsubstrate specificity.

[0115] When used in a detergent, the lipolytic enzyme may also have animproved detergence effect, particularly an improved one-cycle washeffect.

[0116] When used in baking, the lipolytic enzyme may result in a softercrumb and a better elasticity from day 0 to day 7 during storage of thebaked product. Further, loaf volume and standing of the baked productmay be improved. These properties may be evaluated as described in WO0032758.

[0117] Thermostability

[0118] The thermostability can be measured at a relevant pH for theintended application using a suitable buffer. Examples of buffers and pHare: pH 10.0 (50 mM glycine buffer), pH 7.0 (50 mM HEPES Buffer) or pH5.0 (50 mM sodium acetate as buffer).

[0119] For comparison, measurements should be made in the same buffer,at the same conditions and at the same protein concentration. Variousmethods can be used for measuring the thermostability:

[0120] Differential Scanning Calorimetry (DSC)

[0121] In DSC, the heating rate may be 90 degrees per hour. The samplemay be purified to homogeneity, and the melting temperature (T_(M)) maybe taken as an expression of the thermostability.

[0122] Residual enzyme activity

[0123] Alternatively, the thermostability can be determined by measuringresidual lipolytic enzyme activity after incubation at selectedtemperatures. p-nitrophenyl ester in 10 mM Tris-HCl, pH 7.5 may be usedas the substrate, as described in Giver et al., Proc. Natl. Acad. Sci.USA 95(1998)12809-12813 and Moore et al. Nat. Biotech. 14(1996) 458-467.Samples may be added periodically, or only one sample may be used withor without different additives to prevent or enhance denaturing, e.g. ina 96 well format.

[0124] CD spectroscopy

[0125] CD spectroscopy as described e.g. in Yamaguchi et al. Proteinengineering 9(1996)789-795. Typical enzyme concentration is around 1mg/ml, Temperature between 5-80 degrees

[0126] Altered Activity Substrate Specificity

[0127] Compared to the parent lipolytic enzyme, the variant of theinvention may have an altered substrate specificity, i.e. an alteredratio of activities towards different ester bonds in substrates. Thismay be used to increase a desired activity and/or decrease an undesiredactivity and thus decrease the ratio of an undesired activity to adesired activity.

[0128] Thus, an enzyme with increased phospholipase activity may beuseful, e.g., in baking or in purification of vegetable oil. It may bedesired to increase the hydrolytic activity on digalactosyl-diglyceride(DGDG) for use in baking.

[0129] It may be desired to increase the lipase activity for anyindustrial use where lipases are used. For use in detergents or bakingit may be desired to increase the activity on long-chain (C₁₆-C₂₀)triglycerides, and it may be desired to increase the specificity forlong-chain fatty acids by decreasing the ratio of activity onshort-chain or medium-chain (C₄-C₈) fatty acids to the activity onlong-chain fatty acids.

[0130] For use in flavor development in food products (such as cheeseripening) it may be desired to increase the lipase activity onshort-chain or medium-chain (C₄-C₈) triglycerides.

[0131] For use as a phospholipase in purification of vegetable oil, itmay be desired to decrease the ratio of lipase activity on long-chain(C₁₆-C₂₀) triglycerides to the phospholipase activity.

[0132] For use in detergent, the lipolytic enzyme may have an increasedlong-chain/short-chain specificity compared to the parent enzyme, e.g.an increased ratio of activity on long-chain (e.g. C₁₆-C₂₀)triglycerides to the activity on short-chain (e.g. C₄-C₈) triglycerides.This may be determined as the ratio of SLU with olive oil as thesubstrate and LU with tributyrin as substrate (methods described laterin this specification).

[0133] Altered pH Dependence

[0134] The lipolytic enzyme may have an increased pH optimum or adecreased pH optimum.

[0135] The altered pH dependence may be an increased alkaline/neutralactivity ratio, i.e. an increased ratio of lipase activity (e.g. lipaseactivity) at alkaline pH (e.g. pH 9-10) to the activity at neutral pH(around pH 7). This may be determined with tributyrine as the substrateas described later in this specification.

[0136] One-cycle Wash Effect

[0137] The one-cycle wash effect is described in WO 9707202. It may bedetermined by subjecting 7 lard-stained cotton swatches (9×9 cm) perbeaker to a one cycle wash in a thermostated Terg-O-to-Meter (TOM), eachbeaker containing 1000 ml of water comprising 3.2 mM Ca²⁺/Mg²⁺ (in aratio of 5:1) and 5 g/l of said detergent composition, pH 10, andcomprising 12500 LU/I of the lipolytic enzyme, the wash treatment beingcarried out for 20 minutes at a temperature of 30° C., followed byrinsing for 15 minutes in running tap water and overnight line-drying atroom temperature, subsequent extraction and quantification of fattymatter on the swatches by Soxhlet extraction. This may be done usingDetergent Composition A or B described in WO 9707202.

[0138] Use of Variant

[0139] The variants of the invention can be used in known applicationsof lipolytic enzymes by analogy with the prior art, e.g.:

[0140] A variant with lipase activity can be used in the pulp and paperindustry, to remove pitch or to remove ink from used paper. WO 9213130,WO 9207138, JP 2160984 A, EP 374700.

[0141] A variant with phospholipase and/or DGDGase activity can be usedin the preparation of dough, bread and cakes, e.g. to increase doughstability and dough handling properties, or to improve the elasticity ofthe bread or cake. WO 94/04035, WO 00/32758.

[0142] A variant with phospholipase activity can be used in a processfor reducing the content of phospholipid in an edible oil. U.S. Pat. No.5,264,367 (Metallgesellschaft, Röhm); K. Dahlke & H. Buchold, INFORM, 6(12), 1284-91 (1995); H. Buchold, Fat Sci. Technol., 95 (8), 300-304(1993); JP-A 2-153997 (Showa Sangyo); or EP 654,527 (Metallgesellschaft,Röhm).

[0143] A variant with lysophospholipase activity can be used to improvethe filterability of an aqueous solution or slurry of carbohydrateorigin, e.g. starch hydrolysate, especially a wheat starch hydrolysate.EP 219,269.

[0144] A variant with phospholipase activity can be used for thepreparation of lysophospholipid, e.g. lyso-lecithin (EP 870840, JP-A10-42884, JP-A 4-135456 or JP-A 2-49593) of for the production ofmayonnaise (EP 628256, EP 398666 or EP 319064).

[0145] A variant with phospholipase activity may also be used in theprocessing of dairy and other food products, e.g. as described in EP567,662 (Nestlé), EP 426,211 (Unilever), EP 166,284 (Nestlé), JP-A57-189638 (Yakult) or U.S. Pat. No. 4,119,564 (Unilever).

[0146] A variant with activity towards short-chain fatty acyl groups maybe used to release free fatty acids (FFA) for flavor development in foodproducts, e.g. in cheese ripening, e.g. as described in M. Hanson, ZFL,41 (10), 664-666 (1990)).

[0147] A variant with phospholipase activity can be used in the leatherindustry. GB 2233665, EP 505920.

[0148] A variant with lipase activity may be used for removing fattymatter containing hydrophobic esters (e.g. triglycerides) during thefinishing of textiles. WO 93/13256.

[0149] Use in detergent

[0150] The variant may be used as a detergent additive, e.g. at aconcentration (expressed as pure enzyme protein) of 0.001-10 (e.g.0.01-1) mg per gram of detergent or 0.001-100 (e.g. 0.01-10) mg perliter of wash liquor. In detergents, the variant may have a highactivity on long-chain triglycerides (C₁₆-C₂₀) to improve the removal offatty soiling. The variant may have phospholipase activity. The variantmay have low activity towards short-chain (C₄-C₈) fatty acids intriglycerides. WO 97/04079, WO 97/07202, WO 97/41212, WO 98/08939 and WO97/43375.

[0151] The lipolytic enzyme of the invention may be used in a detergentcomposition. It may be included in the detergent composition in the formof a non-dusting granulate, a stabilized liquid, or a protected enzyme.Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.No. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and mayoptionally be coated by methods known in the art. Liquid enzymepreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods.

[0152] The detergent composition may be in any convenient form, e.g. aspowder, granules, paste or liquid. A liquid detergent may be aqueous,typically containing up to 70% water and 0-30% organic solvent, ornon-aqueous.

[0153] The detergent composition comprises one or more surfactants, eachof which may be anionic, nonionic, cationic, or zwitterionic. Thedetergent will usually contain 0-50% of anionic (fatty alcohol sulfate)(AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates(SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinicacid, or soap. It may also contain 0-40% of nonionic surfactant such asalcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, orpolyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).

[0154] The detergent may contain 1-65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates orlayered silicates (e.g. SKS-6 from Hoechst). The detergent may also beunbuilt, i.e. essentially free of detergent builder.

[0155] The detergent may contain a bleaching system which may comprise aH₂O₂ source such as perborate or percarbonate which may be combined witha peracid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfon-ate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of, e.g., the amide, imide, orsulfone type.

[0156] The pH (measured in aqueous solution at use concentration) willusually be neutral or alkaline, e.g. in the range of 7-11.

[0157] Further, the detergent may be a dishwashing detergent withsurfactant typically containing 0-90% of non-ionic surfactant such aslow- to non-foaming ethoxylated propoxylated straight-chain alcohols.

[0158] Nomenclature for Amino Acid Substitutions

[0159] The nomenclature used herein for defining amino acidsubstitutions uses the single-letter code, as described in WO 92/05249.

[0160] Thus, D27N indicates substitution of D in position 27 with N.D27N/R indicates a substitution of D27 with N or R. L227X indicates asubstitution of L227 with any other amino acid. D27N+D111A indicates acombination of the two substitutions.

[0161] Homology and Alignment

[0162] For purposes of the present invention, the degree of homology maybe suitably determined by means of computer programs known in the art,such as GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP withthe following settings for polypeptide sequence comparison: GAP creationpenalty of 3.0 and GAP extension penalty of 0.1.

[0163] In the present invention, corresponding (or homologous) positionsin the lipase sequences of Rhizomucor miehei (rhimi), Rhizopus delemar(rhidl), Thermomyces lanuginosus (former; Humicola lanuginosa) (SP400),Penicillium camembertii (Pcl) and Fusarium oxysporum (FoLnp11), aredefined by the alignment shown in FIG. 1 of WO 00/32758.

[0164] To find the homologous positions in lipase sequences not shown inthe alignment, the sequence of interest is aligned to the sequencesshown in FIG. 1. The new sequence is aligned to the present alignment inFIG. 1 by using the GAP alignment to the most homologous sequence foundby the GAP program. GAP is provided in the GCG program package (ProgramManual for the Wisconsin Package, Version 8, August 1994, GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman,S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-45). The following settings are used for polypeptide sequencecomparison: GAP creation penalty of 3.0 and GAP extension penalty of0.1.

[0165] Procedure for Obtaining Thermostable Variants

[0166] Thermostable variants of a given parent lipolytic enzyme can beobtained by the following standard procedure:

[0167] ξ Mutagenesis (error-prone, doped oligo, spiked oligo)

[0168] ξ Primary Screening

[0169] ξ Identification of more temperature stable mutants

[0170] ξ Maintenance (glycerol culture, LB-Amp plates, Mini-Prep)

[0171] ξ Streaking out on another assay plate—secondary screening (1degree higher then primary screening)

[0172] ξ DNA Sequencing

[0173] ξ Transformation in Aspergillus

[0174] ξ Cultivation in 100 ml scale, purification, DSC

[0175] Primary Screening Assay

[0176] The following assay method is used to screen lipolytic enzymevariants and identify variants with improved thermostability.

[0177]E. coli cells harboring variants of a lipolytic enzyme gene areprepared, e.g. by error-prone PCR, random mutagenesis or localizedrandom mutagenesis or by a combination of beneficial mutants andsaturation mutagenesis.

[0178] The assay is performed with filters on top of a LB agar plate. E.coli cells are grown on cellulose acetate filters supplied withnutrients from the LB agar plate and under the selection pressure ofampicillin supplied with the LB agar. Proteins including the desiredenzyme are collected on a nitrocellulose filter between LB agar andcellulose acetate filter. This nitrocellulose filter is incubated in abuffer of desired pH (generally 6.0) and at the desired temperature for15 minutes (e. g. 78 degrees for the T. lanuginosus lipase). Afterquenching the filters in ice-water, the residual lipase activity isdetermined through the cleavage of indole acetate and the subsequentcoloration of the reaction product with nitro-blue tetrazolium chlorideas described by Kynclova, E et al. (Journal of Molecular Recognition 8(1995)139-145).

[0179] The heat treatment applied is adjusted so that the parentgeneration is slightly active, approximately 5-10% compared to samplesincubated at room temperature. This facilitates the identification ofbeneficial mutants.

1. A lipolytic enzyme which is a variant of a parent fungal lipolyticenzyme, comprising one or more amino acid substitutions corresponding toany of the following in SEQ ID NO: 1: Q15 to A, C, D, E, F, G, I, K, L,M, N, P. R, S, T, V, W, Y; Y16 to A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W; A18 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W, Y; A19 to C, D, E, F, G, H, I, K, L, M, N, Q, R, S, V, W, Y; A20to C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; N25 to A, C, D,E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y; N26 to A, D, E, F, G, H,I, K, L, M, P, Q, R, S, T, V, W, Y; E43 to A, C, D, F, G, H, I, K, L, M,N, R, S, T, V, W, Y; V44 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, W, Y; K46 to A, C, D, E, F, G, H, I, L, M, N, Q, S, T, V, W, Y; A47to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y; A49 to C, D, E,F, G, H, I, K, L, M, N, Q, R, S, V, W, Y; L52 to A, C, D, E, F, G, H, I,K, M, N, P, Q, R, S, T, V, W, Y; Y53 to A, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W; S54 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T,V, W, Y; G65 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;L67 to A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, Y; A68 to C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; L69 to A, C, D, E,F, G, H, I, K, M, N, P, Q, S, T, V, W, Y; T72 to A, C, D, E, F, G, H, I,L, M, N, P, Q, R, S, V, W, Y; K74 to A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, Y; L75 to A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, Y; V77 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W,Y; S79 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y; R81to A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y; S83 to A, C, D,E, F, G, H, I, K, L, M, N, Q, R, V, W, Y; S85 to A, D, E, G, H, I, L, M,N. Q, V, W, Y; W89 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V,Y; L97 to A, C, D, E, F, G, H, I, K, N, P, R, S, T, W, Y; K98 to A, C,G, H, L, M, N, P, Q, S, T, V, W, Y; E99 to C, F, G, I, M, P, W, Y; G106to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y; C107 to A, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; R108 to A, C, D, E,F, G, H, I, K, L, M, N, P, Q, S, T, V, Y; G109 to A, C, D, E, F, H, I,K, L, M, N, P, Q, R, S, T, W, Y; T123 to A, C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, V, W, Y; L124 to A, C, D, E, F, G, H, I, K, M, N, P,Q, R, T. V, W, Y; K127 to A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T,V, W, Y; E129 to A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y;A131 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; V132 toA, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, W, Y; Y138 to A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W; V140 to A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, W, Y; L147 to A, C, D, E, F, G, H, I,K, M, N, P, Q, R, S, T, V, W, Y; A150 to C, D, E, F, G, H, I, K, L, M,N, P, Q, R, S, T, V, W, Y; T153 to A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, Y; Y164 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W; D165 to A, C, E, F, G, H, I, K, L, M, N, Q, S, T, V, W,Y; D167 to A, C, E, F, H, K, L, M, N, P, Q, S, T, V, W, Y; S170 to A, C,D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y; Y171 to A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W; G172 to A, C, D, E, F, H, I,K, L, M, N, P, Q, R, S, T, V, W, Y; A173 to C, D, E, F, G, H, I, K, L,M, N, Q, R, S, T, V, W, Y; P174 to A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, Y; R175 to A, C, D, E, F, G, H, I, K, L, M, N, Q, S,T, V, W, Y; V176 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,W, Y; G177 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R. S, T, V, W, Y;R179 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y; A182 toC, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W. Y; Y194 to A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W; H198 to A, C, D, E,F, G, H, K, L, M , P, Q, R. S, T, V, W, Y; N200 to A, C, D, E, F, G, H,I, K, L, M, P, Q, S, T, V, W, Y; P207 to A, C, D, E, F, G, H, I, K, L,M, N, Q, R, S, T, V, W, Y; P208 to A, C, D, E, F. G, H, I, K, L, M, N,Q, R, S, T, V, W, Y; R209 to C, D, F, G, H, I, K, L, M, N, Q, T, V, W,Y; G212 to A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; S214to A, C, D, E, F, G. H, I, K, L, M, N, P, Q, R, T, V, W, Y; H215 to A,C, D, E, F, G, I, K, L, M, N, P, Q, R, S. T, V, W, Y; S216 to A, C, D,E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y; S217 to A, C, D, E, F, G,H, I, K, L, M, N, P, Q, R, T, V, W, Y; P218 to A, C, D, E, F, G, H, I,K, L, M, N, Q, R, S, T, V, W, Y; E219 to C, D, F, H, I, M, P, W, Y; Y220to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W; K223 to A,C, D, E, F, G, H, I, L, M, N, Q, S, T, V, W, Y; S224 to A, C, D, E, F,G, H, I, K, L, M, N, Q, T, V, W, Y; D234 to C, E, F, H, I, M, W; I235 toA, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y; K237 to A, C,D, E, F, G, H, L, N, P, Q, S, T, V, W, Y; I238 to A, C, D, E, F, G, H,K, L, M, N, P, Q, R, S, T, V, W, Y; D242 to C, E, F, G, H, I, M, P, W,Y; A243 to C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y; P250 toA, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y; P253 to A, C,D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, Y; D254 to C, E, F, H, I,M, P, Y; I255 to C, D, E, F, H, L, M, N, Q, W, Y; P256 to C, E, F, G, H,I, K, L, M, N, Q, R, V, W, Y; Y261 to A, C, E, F, G, H, L, M, N, P, Q,R, S, T, V.
 2. The lipolytic enzyme of any preceding claim wherein theparent lipolytic enzyme has at least 80% homology with SEQ ID NO:
 1. 3.The lipolytic enzyme of the preceding claim wherein the parent lipolyticenzyme is the lipase produced by Thermomyces lanuginosus DSM 4109 andhaving the amino acid sequence of SEQ ID NO:
 1. 4. The lipolytic enzymeof any preceding claim which has one, two, three, four, five or six ofsaid substitutions.
 5. The lipolytic enzyme of any preceding claim whichfurther comprises one or more substitutions of amino acid residues otherthan those listed in claim 1, preferably 1-5 such substitutions.
 6. ADNA sequence encoding the lipolytic enzyme of any preceding claim.
 7. Avector comprising the DNA sequence of the preceding claim.
 8. Atransformed host cell harboring the DNA sequence of claim 6 or thevector of claim
 7. 9. A method of producing the lipolytic enzyme of anyof claims 1-5 comprising a) cultivating the cell of claim 8 so as toexpress and preferably secrete the lipolytic enzyme, and b) recoveringthe lipolytic enzyme.
 10. A method of producing a lipolytic enzymecomprising: a) selecting a parent polypeptide which is a fungallipolytic enzyme, b) preparing a polypeptide derived from the parentpolypeptide by substituting at least one amino acid residuecorresponding to any of the following amino acids in SEQ ID NO: 1 andoptionally substituting one or more other amino acids: Q15, Y16, A18,A19, A20, N25, N26, E43, V44, K46, A47, A49, L52, Y53, S54, G65, L67,A68, L69, T72, K74, L75, V77, S79, R81, S83, S85, W89, D96, L97, K98,E99, G106, C107, R108, G109, T123, L124, K127, E129, A131, V132, Y138,V140, L147, A150, T153, Y164, D165, D167, S170, Y171, G172, A173, P174,R175, V176, G177, R179, A182, Y194, H198, N200, P207, P208, R209, G212,S214, H215, S216, S217, P218, E219, Y220, K223, S224, D234, I235, K237,I238, D242 to A243, P250, P253, D254, I255, P256, Y261, c) testing thelipolytic enzyme activity of the polypeptide, d) selecting a polypeptidehaving lipolytic enzyme activity and an altered property compared to theparent polypeptide, and e) producing the selected polypeptide.
 11. Themethod of the preceding claim wherein the parent polypeptide has atleast 80% homology with SEQ ID NO:
 1. 12. The method of the precedingclaim wherein the parent polypeptide is the lipase produced byThermomyces lanuginosus DSM 4109 and having the amino acid sequence ofSEQ ID NO:
 1. 13. The method of any of claims 10-12 wherein thesubstitutions in step b) comprise substitution corresponding to one ormore of the following in SEQ ID NO: 1: Y16, A18, N25, L52, G65, A68,K74, L75, V77, S79, C107, T123, A131, Y138, L147, A150, T153, Y164,Y171, G172, P174, V176, G177, R179, A182, Y194, H198, P207, P208, G212,S214, H215, S217, P218, Y220, I235, I238, P250.
 14. The method of any ofclaims 10-12 wherein the altered property is an increasedthermostability, an altered pH optimum, or an altered substratespecificity.
 15. A detergent composition comprising the a surfactant andthe lipolytic enzyme of claim 1-5.
 16. A process for preparing a doughor a baked product prepared from the dough which comprises adding thelipolytic enzyme of any of claims 1-5 to the dough, wherein thelipolytic enzyme preferably has phospholipase and/or digalactosyldi-glyceride activity.
 17. The process of claim 16 which furthercomprises adding to the dough an endo-amylase and/or a phospholipid. 18.The process of claim 16 or 17 wherein the endo-amylase is from Bacillus,and is preferably a maltogenic amylase from B. stearothermophilus.